1. Field of the Disclosure
The present invention relates generally to insulin-like growth factor binding proteins (IGFBPs) and, more specifically, to truncated IGFBPs having decreased affinity for IGFs and mitogenic activity.
2. Description of the Related Art
Insulin-like growth factors (IGFs) are low molecular weight polypeptide hormones with structural homology to proinsulin. Two different IGFs are known, namely IGF-I and IGF-II, which are mitogenic in vitro for a wide variety of cells in tissue culture. Both IGFs stimulate in vitro the growth of various tissues and in particular they induce collagen synthesis. IGF-I mediates the growth promoting effect of growth hormone in chondrogenesis and bone formation and is therefore essential for normal growth of an individual. This is demonstrated by the fact that pygmies and toy poodles are deficient in IGF-I but have normal growth hormone level in their serum. IGF-II is believed to play a key role in fetal development and nerve growth.
IGFs stimulate cell growth in a variety of tissues including bone. The growth stimulatory effects of IGF-I and IGF-II in bone have been shown for cells of the osteoblast lineage. Because both IGFs are secreted by this cell type and both are recoverable from bone matrix, they are capable of affecting osteoblast function through autocrine or paracrine mechanisms. Factors that regulate IGF effects in osteoblasts are not completely understood. Regulation of IGF receptor affinity is a well documented mechanism that potentially modulates autocrine-directed cell growth.
Through their association with carrier proteins (hereinafter referred to as IGF binding proteins or IGFBPs), binding of IGFs to cell surface receptors is inhibited. It has also been demonstrated that another function of the IGFBPs is to increase the short half-life of IGFs, which are subjected to rapid proteolytic degradation when present in the free form in blood. IGFs for use in the above-referred treatments are advantageously administered to a subject in association with at least one IGF binding protein.
IGFBPs are secreted by cells in culture and either inhibit or enhance IGF-stimulated functions [Clemmons, D. R., et al., (1991) In Modern Concepts of Insulin-like Growth Factors. E. M. Spencer, editor. Elsevier, New York, N.Y. 475-486]. Known forms of IGFBPs include IGFBP-1, having a molecular weight of approximately 30-40 kd in humans. See, e.g., WO89/09792, published Oct. 19, 1990, Clemmons, D. R., et al., pertaining to cDNA sequences and cloning vectors for IGFBP-1 and IGFBP-2; WO89/08667, published Sep. 21, 1989, Drop, L. S., et al., relating to an amino acid sequence of IGFBP-1; WO89/09268, published Oct. 5, 1989, Baxter, R. C., relating to a cDNA sequence of IGFBP-1 and methods of expression for IGFBP-1.
IGFBP-2 has a molecular weight of approximately 33-36 kd. See, e.g., Binkert, C. et al., The EMBO Journal (1989) 8:2497-2502, relating to a nucleotide and deduced amino acid sequence for IGFBP-2.
IGFBP-3 has a molecular weight of 150 kd. See, e.g., Baxter, R. C. et al., Bioch. Biophys. Res. Com. (1986) 139:1256-1261, pertaining to a 53 kd subunit of IGFBP-3 that was purified from human serum; Wood, W. I. et al., Mol. Endocrinol. (1988) 2:1176-1185, relating to a full length amino acid sequence for IGFBP-3 and cellular expression of the cloned IGFBP-3 CDNA in mammalian tissue culture cells; WO90/00569, published Jan. 25, 1990, Baxter, R. C., relating to isolating from human plasma an acid-labile subunit (ALS) of IGFBP complex and the particular amino acid sequence for ALS pertaining to a subunit of IGFBP-3; Schmid, Ch. et al., Bioch. Biophys. Res Com. (1991) 179:579-585, relates to effects of full length and truncated IGFBP-3 effect on two different osteoblastic cell lines.
Altbough initially some inconsistencies in nomenclature for IGFBP-4, IGFBP-5, and IGFBP-6 existed, in 1991 participants of the 2nd International IGF Symposium agreed upon an accepted IGFBP-4, IGFBP-5, and IGFBP-6 nomenclature. Using accepted terminology, Mohan, S. et al., Proc. Natl. Acad. Sci. (1989) 86:8338-8342, relates to an N-terminal amino acid sequence for an IGFBP-4 isolated from medium conditioned by human osteosarcoma cells, and Shimasaki, S. et al., Mol. Endocrinology (1990) 4:1451-1458, pertains to IGFBP cDNAs encoding IGFBP-4 from rat and human. WO92/03471 published Mar. 5, 1992, Kiefer et al., relates to a new insulin-like growth factor binding protein IGFBP-4 (originally designated therein as IGFBP-5); and WO92/03470 published Mar. 5, 1992, Kiefer et al., relates to genetic material encoding IGFBP-4 (originally designated therein as IGFBP-5).
WO92/12243 published Jul. 23, 1992, Kiefer et al., relates to a new insulin-like growth factor binding protein IGFBP-5 (originally designated therein as IGFBP-6). Andress, D. L. et al., Bioch. Biophys. Res Com. (1991) 176:213-218 relates to the modulation of cellular action of a mixture of affinity-purified IGFBPs from a U-2 cell conditioned media on IGFs.
WO92/03469 published Mar. 5, 1992, Kiefer et al., relates to genetic material encoding IGFBP-6 (originally designated therein as IGFBP-4); and WO92/03152 published Mar. 5, 1992, Kiefer et al., relates to a new insulin-like growth factor IGFBP-6. (originally designated therein as IGFBP-4).
Zapf, J. et al., J. of Biol. Chem. (1990) 265:14892-14898, pertains to four IGFBPs (IGFBP-2, IGFBP-3, a truncated form of IGFBP-3, and IGFBP-4) isolated from adult human serum by insulin-like growth factor (IGF) affinity chromatography and high performance liquid chromatography. Shimasaki, et al., 2nd International IGF Symposium Abstract (January 1991), discussed amino terminal amino acids for IGFBP-4, IGFBP-5, and IGFBP-6.
When administered alone, i.e., without any IGF, the IGFBPs may also be therapeutically useful for blocking the adverse effects of IGFs, such as those which occur when IGFs are produced in excess, e.g. free IGFs secreted by certain cancer cells such as hormone-producing cancer cells such as breast or kidney cancer cells. More recently, it was demonstrated that U-2 human osteosarcoma cells secrete IGFBP-5 and IGFBP-6 [Andress, D. L., and Birnbaum, R. S. (1991) Biochem. Biophys. Res. Commun. 176, 213-218; Shimasaki, S., et al. (1991) J. Biol. Chem. 266, 10646-10653; Shimasaki, S., et al. (1991) Mol. Endocrinol. 5, 938-948]. Although affinity-purified IGF-binding proteins derived from U-2 conditioned medium clearly enhanced IGF-I stimulated mitogenesis [Andress, D. L., and Birnbaum, R. S. (1991) Biochem. Biophys. Res. Commun. 176, 213-218], it was unclear from those studies which protein was responsible for this effect. Mohan et al. demonstrated that IGFBP-4, purified from TE-89 human osteosarcoma cells inhibits IGF-stimulated osteoblast mitogenesis. [Mohan, S., et al. (1989) Proc. Natl. Acad. Sci. (U.S.A.). 86, 8338-8342; see also LaTour, D., et al. (1990) Mol. Endocrinol. 4, 1806-1814].
There is significant interest in the discovery of new properties and applications for known IGFBPs in the presence or absence of IGF.
It has been surprisingly discovered that truncated IGFBPs possess mitogenic and osteogenic activity. This discovery has led to the present invention of promoting, for example, bone growth both in vitro and in vivo. Generally, these truncated IGFBPs exhibit reduced affinity for IGFs. Accordingly, it is an object of the present invention to provide a truncated IGFBP that has a decreased affinity for insulin-like growth factors (IGFs) and/or is capable of stimulating mitogenesis in the presence or absence of exogenous or endogenous IGFs.
The present invention provides truncated IGFBPs using recombinant DNA molecules capable of expressing the truncated IGFBP. and also compounds comprising a truncated IGFBP capable of promoting growth of bone cells, preferably with a pharmaceutically acceptable carrier.
The present invention also provides DNA molecules encoding a truncated IGFBP, which may be used to construct vectors for expression in host systems by recombinant DNA techniques.
Additionally, methods for treating bone disorders and stimulating mitogenic activity in mammals, preferably humans, are provided.